CN103649741B - Evaluate the method for the redox active of nucleic acid molecules and there is the nucleic acid molecules of redox active - Google Patents
Evaluate the method for the redox active of nucleic acid molecules and there is the nucleic acid molecules of redox active Download PDFInfo
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Abstract
The present invention provides the new technique of the redox active that can easily evaluate nucleic acid molecules.The evaluation methodology of the present invention is characterised by, including: detecting step, use the device electrochemically detecting redox reaction, electrochemically detect the redox reaction to substrate being catalyzed by the nucleic acid molecules as evaluation object；And evaluation procedure, by the redox active of nucleic acid molecules described in the evaluation of described redox reaction.As described device, use have possess the substrate of test section, described test section has electrode system, be configured with the device of the described nucleic acid molecules as evaluation object on described substrate.In the present invention, it is configured as the multiple nucleic acids molecule of described evaluation object the most on the substrate, utilizes a device that multiple nucleic acids molecule is evaluated.
Description
Technical field
The present invention relates to the method evaluating the redox active of nucleic acid molecules divide with the nucleic acid with redox active
Son.
Background technology
In the various fields such as clinical treatment, food, environment, need to carry out the detection of target substance.The inspection of above-mentioned target substance
Survey the general interaction utilized with above-mentioned target substance, wherein, the method using the antibody specific binding with above-mentioned target substance
It is used widely.In the method, such as, target substance is made to tie with the tokenized antibody of the oxidoreductasees such as peroxidase
Close.Then, use chromogenic substrate, utilize the above-mentioned enzyme in above-mentioned marking antibody to carry out chromogenic reaction, and its colour developing is carried out
Detection.By the detection of above-mentioned colour developing, indirectly carry out the analysis of above-mentioned target substance, such as qualitative analysis and quantitative analysis.
But, above-mentioned antibody obtains, therefore, for toxicity target substance and low molecule by animal is carried out immunity
It is extremely difficult that target substance obtains specific antigen.Therefore, in recent years, the nucleic acid being combined with target substance, the most so-called nucleic acid
Fit (the most fit) receives publicity.Above-mentioned fit can in vitro obtain, therefore, for such as toxicity target substance and
Low molecule target substance also is able to obtain fit.And, attempt such above-mentioned antibody of fit replacement is being used for target substance
During detection and with demonstrate as peroxidase catalysis activity DNAzyme (DNAzyme).In general, above-mentioned de-
Oxygen ribozyme is to have the structural motif rich in guanine, in G-tetra-stranded structure, by being combined formation with hemin again
Fit and play the DNA of the catalysis of peroxidase.
In the detection of above-mentioned target substance, specifically, the list being formed by connecting by aptamer is utilized with strand DNAzyme
Chain nucleic acid elements (non-patent literature 1).In the case of there is not target substance, above-mentioned single-chain nucleic acid element passes through self annealing shape
Become stem structure, make due to above-mentioned stem structure above-mentioned DNAzyme become can not in G-the structure of tetra-serobilas.Therefore, do not exist
Under conditions of target substance, the above-mentioned DNAzyme in above-mentioned single-chain nucleic acid element can not be combined with hemin, it is impossible to sends out
Wave catalysis.On the other hand, in the presence of target substance, above-mentioned target substance and above-mentioned fit combination, thus make above-mentioned strand core
Acid element eliminates above-mentioned stem structure.Therefore, the above-mentioned DNAzyme shape in the presence of target substance, in above-mentioned single-chain nucleic acid element
Become G-tetra-serobila, be combined with hemin, thus play above-mentioned catalysis.Therefore, alive by making for oxidoreduction
Property chromogenic substrate coexist, when there is above-mentioned target substance occur chromogenic reaction, do not show when there is not above-mentioned target substance
Colour response.Therefore, by the detection of chromogenic reaction, it is possible to carry out the analysis of target substance.Also, it is not necessary to target substance is marked
Note, therefore, it is possible to directly detect using the extensive target substance comprising low molecule as object.
So, above-mentioned single-chain nucleic acid element needs to be controlled the work of above-mentioned DNAzyme by above-mentioned fit stereochemical structure
Property.It is thus possible, for instance, it is desirable to the fit sequence according to being used carrys out the DNAzyme that combined activity is easily controlled.
But, the limited amount of the DNAzyme being in the news.Accordingly, it is determined that during with the fit combination of regulation, it has to
Select from limited DNAzyme, be restricted according to the nucleic acid elements that target substance builds precision more excellent.It addition, in order to enable
Enough carry out the detection that sensitivity is more excellent, need to show the DNAzyme of either high redox activity.
Therefore, attempted the DNAzyme that acquisition is new, but in the screening of DNAzyme, can only be for candidate nucleic acid molecules one
An individual ground confirms its activity, and its operation is the most loaded down with trivial details.
Prior art literature
Non-patent literature
Non-patent literature 1:Teller etc., Anal.Chem., 2009, vol.81, p.9114-9119
Summary of the invention
Invent problem to be solved
Therefore, present invention aim at providing the new technique of the redox active that can easily evaluate nucleic acid molecules.
For the method solving problem
The evaluation methodology of the present invention is the method for the redox active evaluating nucleic acid molecules, it is characterised in that including: inspection
Survey step, use the device electrochemically detecting redox reaction, electrochemically detect by the core as evaluation object
The redox reaction to substrate of acid molecule catalysis and evaluation procedure, by the evaluation of above-mentioned redox reaction
The redox active of above-mentioned nucleic acid molecules, and, said apparatus has the substrate possessing test section, and above-mentioned test section has electricity
Electrode systems, aforesaid substrate is configured with the above-mentioned nucleic acid molecules as evaluation object.
The nucleic acid molecules of the present invention is the nucleic acid molecules with redox active, it is characterised in that containing selecting free sequence
At least one polynucleotide in the group of row number 1～132 composition.
Invention effect
Evaluation methodology according to the present invention, it is possible to easily evaluate oxidoreduction for the nucleic acid molecules as evaluation object
The presence or absence of activity and intensity thereof.It addition, according to the evaluation methodology of the present invention, such as, additionally it is possible to multiple nucleic acids molecule is entered simultaneously
Row is evaluated, therefore, it is possible to screening purpose nucleic acid molecules efficiently.As it has been described above, there is the nucleic acid molecules of redox active such as
The enzymes such as peroxidase can be replaced to use, therefore, useful in the various fields such as clinical treatment, food, environment.
Accompanying drawing explanation
Fig. 1 is that the spacer lengths represented in embodiments of the invention 1 on pH, electrode is different with concentration of hydrogen peroxide
Under the conditions of the figure of the signal of telecommunication that produced by DNAzyme, (A) is the result of pH7.4, and (B) is the result of pH8.0, and (C) is pH8.5
Result, (D) is the result of pH9.0.
Fig. 2 is the figure representing the signal of telecommunication produced by DNAzyme in embodiments of the invention 1, and (A) is to represent identical
The first time signal of telecommunication of microarray measure the figure of the repeatability measured with the second time signal of telecommunication, (B) is to represent identical interval
The figure of the repeatability between the DNAzyme of section length.
Fig. 3 is the relation representing the value of electrical signals produced by each DNAzyme in embodiments of the invention 1 with its frequency
Figure.
Fig. 4 is the figure representing the signal of telecommunication produced by DNAzyme in embodiments of the invention 2, and (A) is to represent identical
The first time signal of telecommunication of microarray measure the figure of the repeatability measured with the second time signal of telecommunication, (B) is to represent identical micro-battle array
The second time signal of telecommunication of row measures the figure of the repeatability measured with the third time signal of telecommunication, and (C) is represent identical microarray the
Signal of telecommunication measures the figure of the repeatability measured with the third time signal of telecommunication.
Fig. 5 is the relation representing the value of electrical signals produced by each DNAzyme in embodiments of the invention 2 with its frequency
Figure, (A) represent c-Myc result, (B) represent SA result, (C) represent EAD2 result, (D) represent TA result.
Fig. 6 is the figure representing the value of electrical signals produced by each DNAzyme in embodiments of the invention 3.
Detailed description of the invention
1. evaluation methodology
As it has been described above, the evaluation methodology of the present invention is characterised by, including: detecting step, use and electrochemically examine
Survey the device of redox reaction, electrochemically detect the oxidation to substrate being catalyzed by the nucleic acid molecules as evaluation object also
Former reaction and
Evaluation procedure, by the redox active of the above-mentioned nucleic acid molecules of the evaluation of above-mentioned redox reaction,
Said apparatus has the substrate possessing test section,
Above-mentioned test section has electrode system,
The above-mentioned nucleic acid molecules as evaluation object it is configured with on aforesaid substrate.
The above-mentioned nucleic acid molecules as evaluation object can be such as a kind of, the most multiple.In the case of the latter, example
As, by configuring multiple nucleic acids molecule on aforesaid substrate, it is possible to use a substrate that above-mentioned multiple nucleic acids molecule is commented
Valency.
The above-mentioned nucleic acid molecules as evaluation object for example, contains the molecule of nucleotide residue.Above-mentioned nucleic acid molecules example
As being the molecule being only made up of nucleotide residue, it is also possible to for the molecule containing nucleotide residue.Above-mentioned nucleotide is such as
For ribonucleotide, deoxyribonucleotide and their derivant.Above-mentioned nucleic acid molecules such as can contain only ribonucleotide
Any one in acid, deoxyribonucleotide and their derivant, it is also possible to containing more than two kinds, it is also possible to all contain
Have.Specifically, above-mentioned nucleic acid molecules can be such as the RNA containing ribonucleotide and/or its derivant, it is also possible to for containing
There is the DNA of deoxyribonucleotide and/or its derivant, it is also possible to for containing the former and the chimera (DNA/RNA) of the latter.On
Stating nucleic acid molecules can be strand, it is also possible to for double-strand, preferably strand.In the case of above-mentioned nucleic acid molecules is strand, above-mentioned
Nucleic acid molecules can be enumerated such as: single stranded DNA, single stranded RNA, single-chain chimeric body (DNA/RNA) etc., is double at above-mentioned nucleic acid molecules
In the case of chain, above-mentioned nucleic acid molecules can be enumerated such as: double-stranded DNA, double-stranded RNA, the double-strand of DNA-RNA, double-strand chimeric
Body (DNA/RNA) etc..The length of above-mentioned nucleic acid molecules is not particularly limited, and for example, 11～80 bases are long.
For above-mentioned nucleotide, as base, the most natural base (unartificial base), non-natural alkali can be contained
Base (artificial bases).Above-mentioned natural base can be enumerated such as: A, C, G, T, U and their modified base.Above-mentioned modification is permissible
Enumerate such as: methylate, be fluorinated, amination, sulfuration etc..Above-mentioned nonnatural base can be enumerated such as: 2 '-fluoropyrimidine, 2 '-O-
Methylpyrimidines etc., as concrete example, can enumerate: 2 '-fluorouracil, 2 '-amino-uracil, 2 '-O-methyluracils, 2-sulfur
Uracil etc..Above-mentioned nucleotide can be such as the nucleotide after modifying, and above-mentioned modified nucleotide can be enumerated such as: 2 '-first
Base-uridylate residue, 2 '-methylated-cytosine nucleotide residue, 2 '-fluorination-uridylate residue, 2 '-
Fluorination-cytidylic acid residue, 2 '-amination-uridylate residue, 2 '-amination-cytidylic acid residue,
2 '-sulfuration-uridylate residue, 2 '-sulfuration-cytidylic acid residue etc..Above-mentioned nucleic acid molecules can comprise such as
PNA (peptide nucleic acid(PNA)), LNA (Locked Nucleic Acid locks nucleotide) etc..
In the present invention, as long as above-mentioned redox reaction is such as during substrate generation product, at the bottom of two kinds
The reaction given and accepted of electronics is there is between thing.The kind of above-mentioned redox reaction is not particularly limited.It is catalyzed above-mentioned oxygen
Change the activity of reduction reaction and can enumerate such as activity as enzyme, specifically, can enumerate such as with peroxidase
Same activity (hereinafter referred to as peroxidase sample activity) etc..Above-mentioned peroxidase activity can enumerate such as derive from peppery
Peroxidase (HRP) activity of root.It is DNA at the above-mentioned nucleic acid molecules as evaluation object and there is the work of above-mentioned oxidoreduction
In the case of property, above-mentioned DNA is properly termed as such as DNA enzymatic or DNAzyme, it addition, be RNA at above-mentioned nucleic acid molecules and have
In the case of above-mentioned redox active, above-mentioned RNA is properly termed as such as RNase or ribozyme (RNAzyme).
In the present invention, as long as the above-mentioned test section of said apparatus can detect by the above-mentioned nucleic acid molecules as evaluation object
The signal of telecommunication that the redox reaction being catalyzed produces.As it has been described above, above-mentioned test section has above-mentioned electrode system.Above-mentioned
Electrode system such as can comprise working electrode and to electrode, it is also possible to comprises working electrode, to electrode and reference electrode.Above-mentioned
The material of electrode is not particularly limited, and can enumerate such as: platinum, silver, gold, carbon etc..Above-mentioned working electrode and above-mentioned can to electrode
To enumerate such as: platinum electrode, silver electrode, gold electrode, carbon electrode etc., above-mentioned reference electrode can be enumerated such as: silver/silver chloride electricity
Pole etc..Above-mentioned silver/silver chloride electrode such as can be formed by stacking silver chloride electrode on silver electrode.
Above-mentioned test section such as can be formed by configuring above-mentioned electrode on the surface of aforesaid substrate.Above-mentioned electrode
Collocation method is not particularly limited, and can use such as known method, and concrete example can enumerate vapour deposition method, sputtering method, silk screen
The film forming method such as print process, galvanoplastic.Above-mentioned electrode such as can directly be arranged on aforesaid substrate, it is also possible to indirectly
It is arranged on aforesaid substrate.Indirectly configuration can be enumerated such as across the configuration of miscellaneous part.
Aforesaid substrate is not particularly limited, and such as preferred surface is the substrate of insulating properties.Aforesaid substrate can be such as bag
Substrate containing insulant or the substrate being made up of above-mentioned insulant, it is also possible to there is the insulation comprising insulant for surface
Layer or the substrate of insulating barrier being made up of above-mentioned insulant.Above-mentioned insulant is not particularly limited, and can enumerate such as: glass
The known material such as glass, pottery, insulated plastic, paper.Above-mentioned insulated plastic is not particularly limited, and can enumerate such as: have
Machine silicones, polyimide resin, epoxy resin, fluororesin etc..
As it has been described above, the above-mentioned nucleic acid molecules as evaluation object can be a kind of, but the most multiple, specifically,
On aforesaid substrate, preferably configure above-mentioned multiple nucleic acids molecule.Said apparatus is the most preferably configured with above-mentioned many on aforesaid substrate
Plant the microarray of nucleic acid molecules.Above-mentioned multiple nucleic acids molecule is the most preferably configured to rectangular on aforesaid substrate.It addition, in order to
Can detect, according to its kind, the redox reaction produced by above-mentioned nucleic acid molecules respectively, said apparatus the most preferably has many
Individual test section, and it is each configured with different types of above-mentioned nucleic acid molecules at each test section.Specifically, said apparatus is such as
Can be formed, i.e. by the surface of aforesaid substrate being divided into matrix, forming electrode as above in each zoning
System configures above-mentioned nucleic acid molecules as test section and at each test section.Alternatively, it is also possible to by using commercially available electrochemistry to examine
Survey is microarray, makes the above-mentioned nucleic acid molecules as evaluation object probe on above-mentioned array be combined and is used as above-mentioned evaluation use
Device uses.Above-mentioned commercially available microarray can enumerate such as trade name CombiMatrix ElectraSense
The microarray (CombiMatrix company) etc. of microarray.
It is as it has been described above, if the above-mentioned nucleic acid molecules as evaluation object is arranged on aforesaid substrate, the most fixing
On aforesaid substrate.Above-mentioned nucleic acid molecules such as can directly be arranged on aforesaid substrate, it is also possible to is indirectly configuring at
State on substrate.Specifically, above-mentioned nucleic acid molecules is such as preferably configured on the above-mentioned test section in aforesaid substrate, more preferably joins
Put on the above-mentioned electrode in above-mentioned test section, be preferably configured on the above-mentioned working electrode in above-mentioned electrode.Above-mentioned nucleic acid divides
Son such as can directly be arranged on above-mentioned test section or above-mentioned electrode, it is also possible to be indirectly configuring at above-mentioned test section or on
State on electrode.Hereinafter, unless otherwise noted, then " above-mentioned nucleic acid molecules is to the configuration of aforesaid substrate or immobilization " also comprise upwards
State the above-mentioned test section in substrate or the configuration of the above-mentioned electrode in above-mentioned test section or immobilized implication.
The collocation method of above-mentioned nucleic acid molecules is not particularly limited, and can use known DNA immobilization method.Above-mentioned
Process for fixation can be enumerated and is such as fixed on aforesaid substrate by pre-prepd nucleic acid molecules, is preferably fastened to above-mentioned detection
The method in portion, being more preferably fixed on above-mentioned electrode.The method for example, utilizes photolithographic method, as concrete example, and can
With with reference to No. 5424186 description of United States Patent (USP) etc..It addition, above-mentioned process for fixation can enumerate such as on aforesaid substrate,
Preferably on above-mentioned test section, the more preferably method of nucleic acid on above-mentioned electrode.The method can be enumerated the most so-called
Spotting method (spot method), as concrete example, is referred to United States Patent (USP) No. 5807522 description, Japanese Kohyo 10-
No. 503841 publications etc..
Above-mentioned nucleic acid molecules such as can be fixed on above-mentioned with any one end side in 5 ' end sides or 3 ' end sides
On substrate.
Above-mentioned nucleic acid molecules is arranged on aforesaid substrate preferably for example by bonding pad.Above-mentioned bonding pad such as preferably comprises
Nucleotide residue.Above-mentioned bonding pad such as can be only made up of nucleotide residue, it is also possible to containing nucleotide residue.Above-mentioned nucleoside
Acid is as previously mentioned.The length of above-mentioned bonding pad is not particularly limited, and for example, 1～60 base is long, preferably 6～60 bases
Long, more preferably 20～30 bases are long.Bonding pad is such as also referred to as spacer.
In said apparatus, the above-mentioned nucleic acid molecules as evaluation object can be such as to connect to have fit state.Hereinafter,
The above-mentioned nucleic acid molecules as evaluation object and the above-mentioned fit material being formed by connecting are referred to as nucleic acid elements.Above-mentioned fit do not have
Limiting especially, for example, can be combined with specific target substance is fit.Above-mentioned fit being such as preferably contains nucleotide residue
Nucleic acid molecules as constituent.Above-mentioned nucleotide is not particularly limited, as previously mentioned.Above-mentioned can be combined with target substance
Fit such as can pass through known SELEX (Systematic Evolution of Ligands by Exponential
Enrichment, index concentration Fas lignand system is evolved) method etc. manufactures.Above-mentioned fit can be such as strand, it is also possible to for double
Chain, preferably strand.
Above-mentioned nucleic acid molecules with above-mentioned fit such as can be directly in conjunction with, it is also possible to indirectly combine.In the case of the latter,
It is above-mentioned that both such as can be combined by bonding pad.Above-mentioned nucleic acid molecules with above-mentioned fit such as can with 5 ' ends of one with
3 ' ends of another one connect, it is also possible to connect between both 5 ' ends or between 3 ' ends.
On above-mentioned nucleic acid molecules connect have above-mentioned fit in the case of, an end of the most preferred above-mentioned nucleic acid molecules
Be combined with aforesaid substrate, another end and above-mentioned fit combination.As it has been described above, between aforesaid substrate and above-mentioned nucleic acid molecules,
Above-mentioned nucleic acid molecules and above-mentioned fit between such as can be combined by bonding pad respectively.For above-mentioned nucleic acid molecules with above-mentioned
For fit, such as preferably single stranded nucleic acid molecule is connected with aptamer.The above-mentioned above-mentioned nucleic acid elements example that both are formed by connecting
As stem structure and/or ring structure can be formed by self annealing.
About the evaluation methodology of the present invention, as the first embodiment, illustrate use and be configured with above-mentioned as evaluation object
The method of device of nucleic acid molecules, as the second embodiment, illustrate to use to be configured with and be combined with above-mentioned fit above-mentioned work
For the device of nucleic acid molecules of evaluation object, the method that is i.e. configured with the device of above-mentioned nucleic acid elements.The invention is not restricted to these
Embodiment.
(the first embodiment)
In first embodiment of the present invention, as it has been described above, use be configured with on aforesaid substrate above-mentioned as evaluate right
The device of the nucleic acid molecules of elephant.
First, in above-mentioned detecting step, such as, in the presence of above-mentioned substrate, electrochemically detect by above-mentioned nucleic acid
The redox reaction of Journal of Molecular Catalysis.In the case of above-mentioned nucleic acid molecules has above-mentioned redox active, by above-mentioned core
Acid molecule, such as, generates product from above-mentioned substrate, giving and accepting of electronics occurs in this process.This electronics is given and accepted and such as can be led to
Cross and be applied on electrode electrochemically detect in the above-mentioned test section of said apparatus as electronic signals.Above-mentioned electricity
The detection of signal such as can be carried out by the intensity measuring the above-said current signal such as electric current.
Above-mentioned substrate such as can supply externally to the above-mentioned nucleic acid molecules in said apparatus in above-mentioned detecting step.
Above-mentioned substrate is not particularly limited, and can enumerate such as: hydrogen peroxide, TMB
(TMB), 1,2-phenylenediamine (OPD), double (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts (ABTS) of 2,2 '-azino, 3,3 '-
Diaminobenzidine (DAB), 3,3 '-diaminobenzidine four hydrochlorate (DAB4HCl), AEC (AEC),
4-chloro-1-naphthols (4C1N), 2,4,6-tri-bromo-3-hydroxy benzoic acid, 2,4 dichloro phenol, 4-AA, 4-amino
Phenazone hydrochlorate, luminol etc..
In above-mentioned detecting step, in addition to above-mentioned substrate, such as can coexist porphyrin.In known DNAzyme
Exist and such as demonstrate the DNAzyme of higher redox active by forming complex with porphyrin.Therefore, the present invention
In, for example, it is also possible to detected as the redox active with the complex of porphyrin by the porphyrin that coexists.
Above-mentioned porphyrin is not particularly limited, and can enumerate the most unsubstituted porphyrin, its derivant.Said derivative is permissible
Enumerate the most substituted porphyrin and form complex and the metalloporphyrin etc. that obtains with metallic element.Above-mentioned substituted porphyrin can
To enumerate such as N-methyl Mesoporphyrin etc..It is blood red that above-mentioned metalloporphyrin can enumerate such as chlorine high ferro as ferric complex
Element etc..The most preferred above-mentioned metalloporphyrin of above-mentioned porphyrin, more preferably hemin.
In above-mentioned detecting step, the condition carrying out above-mentioned redox reaction is not particularly limited.PH for example, 7.4～
9.0, preferably 7.4～8.5, more preferably 7.4.Above-mentioned pH is such as controlled preferably by buffer, it is, for example possible to use
The buffer such as the Tris-HCl showing above-mentioned pH.The form that above-mentioned substrate is such as preferably mixing in buffer with substrate solution adds
It is added in above-mentioned nucleic acid molecules.The concentration of the above-mentioned substrate in above-mentioned substrate solution is not particularly limited, for example, 10～
200mmol/L, preferably 20～100mmol/L, more preferably 40～60mmol/L, particularly preferably 50mmol/L.
Then, based on the testing result in above-mentioned detecting step, in above-mentioned evaluation procedure, above-mentioned nucleic acid molecules is evaluated
Redox active.Above-mentioned evaluation procedure such as can evaluate the presence or absence of redox active, it is also possible to evaluates above-mentioned oxidation also
The intensity of former activity.In the case of the latter, such as, can evaluate on the basis of the redox active of arbitrary nucleic acid molecules
The intensity of relative activity, above-mentioned arbitrary nucleic acid molecules the most preferably shows the nucleic acid molecules of redox active.
(the second embodiment)
In second embodiment of the present invention, as it has been described above, use be configured with on aforesaid substrate be combined with above-mentioned fit
The device of the above-mentioned nucleic acid molecules as evaluation object.Unless otherwise noted, then as above-mentioned first embodiment.
First, in above-mentioned detecting step, such as, in the presence of above-mentioned substrate and above-mentioned target substance, electrochemically examine
Survey the redox reaction being catalyzed by above-mentioned nucleic acid molecules.Above-mentioned detecting step is particularly preferably included in and there is above-mentioned substrate and not
The step of above-mentioned redox reaction and depositing in above-mentioned substrate and above-mentioned target substance is detected under conditions of there is above-mentioned target substance
Step at the above-mentioned redox reaction of lower detection.For there is not detection under conditions of above-mentioned target substance and there is above-mentioned target
Detection under conditions of material, first carrying out which kind of detection can.Such as, it is not necessary to make the target substance with above-mentioned fit combination take off
From, it is therefore preferable that the most there is not the detection under conditions of above-mentioned target substance.
The detection that there is not detection under conditions of above-mentioned target substance and exist under conditions of above-mentioned target substance former
Because of as follows.In the detection of target substance, using can be combined with above-mentioned target substance fit and there is the core of redox active
In the case of acid molecule, it is desirable to the above-mentioned nucleic acid molecules with redox active the most only shows under conditions of there is target substance
Show activity, under conditions of there is not target substance, do not show activity.If it is this is because aobvious under conditions of there is not target substance
Show activity, then can obtain false-positive result in target substance detects.Therefore, under conditions of there is not above-mentioned target substance with or without
Redox active and in the presence of above-mentioned target substance with or without redox active, for using above-mentioned fit target quality inspection
Survey is established electrochemical method extremely important.
It addition, do not show activity under conditions of there is not above-mentioned target substance at above-mentioned nucleic acid molecules, in above-mentioned target substance
In the presence of show activity in the case of, it is believed that above-mentioned nucleic acid molecules and above-mentioned fit between set up such as the following stated
Relation in structure.Fit generally by identifying that target substance combination make its stereochemical structure change.Therefore, there is not target
Fit stereochemical structure under conditions of material makes above-mentioned nucleic acid molecules in connection be the structure that activity is closed, the opposing party
Face, if the above-mentioned fit stereochemical structure changed by the combination with target substance makes the suppression of the activity of above-mentioned nucleic acid molecules
It is released from, the most only under conditions of target substance exists, the activity unlatching of above-mentioned nucleic acid molecules.
In above-mentioned detecting step, above-mentioned substrate and above-mentioned target substance such as can above-mentioned externally in said apparatus
Nucleic acid molecules supplies.Above-mentioned substrate is not particularly limited with the order of addition of above-mentioned target substance, for example, it is possible to after adding one
Add another one, it is also possible to add both simultaneously.The above-mentioned porphin furthermore it is possible to coexisted in the same manner as above-mentioned first embodiment
Quinoline.
Above-mentioned substrate is not particularly limited, as aforementioned.Above-mentioned target substance fit is not particularly limited with above-mentioned, permissible
Use desired target substance and can be in connection fit.
Then, in above-mentioned evaluation procedure, based on the testing result in above-mentioned detecting step, above-mentioned nucleic acid molecules is evaluated
Redox active.In above-mentioned evaluation procedure, for example, it is possible to evaluate the presence or absence of redox active, it is also possible to evaluate above-mentioned oxygen
Change the intensity of reducing activity.In the case of the latter, for example, it is possible to come on the basis of the redox active of arbitrary nucleic acid molecules
Evaluating the intensity of relative activity, above-mentioned arbitrary nucleic acid molecules the most preferably shows the nucleic acid molecules of redox active.
In above-mentioned evaluation procedure, such as, do not show above-mentioned oxidation also preferably under conditions of there is not above-mentioned target substance
The nucleic acid molecules of former activity, evaluates the above-mentioned redox active in the presence of above-mentioned target substance.In above-mentioned evaluation procedure, example
As, the presence or absence of redox active can be evaluated, it is also possible to evaluate the intensity of above-mentioned redox active.In the case of the latter,
For example, it is possible to evaluate relative activity on the basis of the redox active of the arbitrary nucleic acid molecules of display redox active
Intensity.
2. screening technique
As it has been described above, the screening technique that the screening technique of the present invention is a kind of nucleic acid molecules with redox active,
It is characterized in that, according to the evaluation methodology of the invention described above, use device to the oxidation of the nucleic acid molecules as evaluation object also
Former activity is evaluated, and screening has the nucleic acid molecules of redox active.
The feature of the screening technique of the present invention is that the evaluation methodology according to the invention described above is evaluated above-mentioned as evaluation object
The redox active of nucleic acid molecules, other steps and condition do not have any restriction.
In the present invention, evaluation result based on above-mentioned redox active.Such as can select to show redox active
Nucleic acid molecules, and then the nucleic acid molecules showing relatively strong redox active can be selected as purpose nucleic acid molecules.Separately
Outward, as it has been described above, such as can also select not show activity under conditions of there is not above-mentioned target substance, deposit in above-mentioned target substance
Show that the nucleic acid molecules of activity is as purpose nucleic acid molecules lower.
3. there is the nucleic acid molecules of redox active
The nucleic acid molecules with redox active of the present invention is characterised by, comprises choosing freely following (a)～(d) group
At least one polynucleotide in the group become.
A polynucleotide that () is made up of any one base sequence in serial number 1～132；
B () is by lacking, replace, insert and/or add one or more bases in above-mentioned (a) in any one base sequence
After base sequence constitute and have the polynucleotide of redox active；
C () is made up of the base sequence of the homogeneity having more than 80% relative to any one base sequence in above-mentioned (a)
And there are the polynucleotide of redox active；
D () is by many with the multi-nucleotide hybrid being made up of any one base sequence in above-mentioned (a) under strict conditions
The complementary base sequence of nucleotide constitutes and has the polynucleotide of redox active.
The nucleic acid molecules of the present invention can be such as be made up of any one polynucleotide in above-mentioned (a)～(d) point
Son, it is also possible to for the molecule containing above-mentioned polynucleotide.In the case of the latter, such as, as described later, the nucleic acid molecules of the present invention
Can be containing the polynucleotide in above-mentioned (a)～(d) of more than 2.The polynucleotide of above-mentioned more than 2 can be identical sequence
Row, it is also possible to for different sequences.It addition, in the case of the latter, the nucleic acid molecules of the present invention can have further and such as connects
Connect district and/or appended sequence etc..The nucleic acid molecules of the present invention is also referred to as DNAzyme.
The polynucleotide of above-mentioned (a) are the many nucleoside being made up of any one base sequence in above-mentioned serial number 1～132
Acid.
Table 1
| Serial number: | Title | Sequence |
| 1 | c0984 | TGAGGGCCGGGTGGGTCGGGAA |
| 2 | c0568 | TGAGGGGAGGGCGGGTCGGGAA |
| 3 | c0067 | TGAGGGATGGGAGGGAGGGGAA |
| 4 | c0192 | TGAGGGAGGGGCGGGCCGGGAA |
| 5 | c0524 | TGAGGGGAGGGAGGGGCGGGAA |
| 6 | c0451 | TGAGGGTCGGGAGGGAGGGGAA |
| 7 | c0541 | TGAGGGGAGGGTGGGCAGGGAA |
| 8 | c0629 | TGAGGGGTGGGCGGGTAGGGAA |
| 9 | c0184 | TGAGGGAGGGGCGGGTCGGGAA |
| 10 | c0760 | TGAGGGGCGGGCGGGTCGGGAA |
| 11 | c0728 | TGAGGGGCGGGTGGGTCGGGAA |
| 12 | e0064 | CTGGGCGGGCGGGCGGGA |
| 13 | c0719 | TGAGGGGCGGGAGGGCGGGGAA |
| 14 | c0531 | TGAGGGGAGGGTGGGAGGGGAA |
| 15 | c0711 | TGAGGGGCGGGAGGGTGGGGAA |
| 16 | c0096 | TGAGGGATGGGTGGGCCGGGAA |
| 17 | c0595 | TGAGGGGTGGGTGGGAGGGGAA |
| 18 | c0335 | TGAGGGTTGGGAGGGCGGGGAA |
| 19 | c0456 | TGAGGGTCGGGAGGGTCGGGAA |
| 20 | c0756 | TGAGGGGCGGGCGGGACGGGAA |
| 21 | c0717 | TGAGGGGCGGGAGGGCAGGGAA |
| 22 | c0712 | TGAGGGGCGGGAGGGTCGGGAA |
| 23 | c0562 | TGAGGGGAGGGCGGGATGGGAA |
| 24 | c0713 | TGAGGGGCGGGAGGGGAGGGAA |
| 25 | c0735 | TGAGGGGCGGGTGGGCGGGGAA |
| 26 | e0021 | CTGGGTGGGTGGGAGGGA |
| 27 | c0607 | TGAGGGGTGGGTGGGCGGGGAA |
| 28 | c0722 | TGAGGGGCGGGTGGGATGGGAA |
| 29 | c0600 | TGAGGGGTGGGTGGGTCGGGAA |
| 30 | e0032 | CTGGGTGGGCGGGCGGGA |
| 31 | c0544 | TGAGGGGAGGGTGGGCCGGGAA |
| 32 | c0455 | TGAGGGTCGGGAGGGTGGGGAA |
| 33 | c0605 | TGAGGGGTGGGTGGGCAGGGAA |
| 34 | c0718 | TGAGGGGCGGGAGGGCTGGGAA |
| 35 | c0586 | TGAGGGGTGGGAGGGGTGGGAA |
| 36 | c0344 | TGAGGGTTGGGTGGGTCGGGAA |
| 37 | c0152 | TGAGGGAGGGGTGGGTCGGGAA |
| 38 | c0630 | TGAGGGGTGGGCGGGTTGGGAA |
| 39 | c0632 | TGAGGGGTGGGCGGGTCGGGAA |
| 40 | c0626 | TGAGGGGTGGGCGGGATGGGAA |
Table 2
| Serial number: | Title | Sequence |
| 41 | c0762 | TGAGGGGCGGGCGGGGTGGGAA |
| 42 | c0707 | TGAGGGGCGGGAGGGAGGGGAA |
| 43 | c0759 | TGAGGGGCGGGCGGGTGGGGAA |
| 44 | c0543 | TGAGGGGAGGGTGGGCGGGGAA |
| 45 | c0637 | TGAGGGGTGGGCGGGCAGGGAA |
| 46 | c0606 | TGAGGGGTGGGTGGGCTGGGAA |
| 47 | c0895 | TGAGGGCTGGGCGGGCGGGGAA |
| 48 | c0753 | TGAGGGGCGGGCGGGAAGGGAA |
| 49 | c0625 | TGAGGGGTGGGCGGGAAGGGAA |
| 50 | c0584 | TGAGGGGTGGGAGGGTCGGGAA |
| 51 | c0567 | TGAGGGGAGGGCGGGTGGGGAA |
| 52 | c0599 | TGAGGGGTGGGTGGGTGGGGAA |
| 53 | c0520 | TGAGGGGAGGGAGGGTCGGGAA |
| 54 | c0636 | TGAGGGGTGGGCGGGGCGGGAA |
| 55 | c0627 | TGAGGGGTGGGCGGGAGGGGAA |
| 56 | c0519 | TGAGGGGAGGGAGGGTGGGGAA |
| 57 | c0343 | TGAGGGTTGGGTGGGTGGGGAA |
| 58 | c0628 | TGAGGGGTGGGCGGGACGGGAA |
| 59 | c0383 | TGAGGGTTGGGCGGGCGGGGAA |
| 60 | c0856 | TGAGGGCTGGGTGGGTCGGGAA |
| 61 | c0709 | TGAGGGGCGGGAGGGTAGGGAA |
| 62 | c0574 | TGAGGGGAGGGCGGGCTGGGAA |
| 63 | c0842 | TGAGGGCTGGGAGGGGTGGGAA |
| 64 | c0638 | TGAGGGGTGGGCGGGCTGGGAA |
| 65 | c0583 | TGAGGGGTGGGAGGGTGGGGAA |
| 66 | c0472 | TGAGGGTCGGGTGGGTCGGGAA |
| 67 | c0463 | TGAGGGTCGGGAGGGCGGGGAA |
| 68 | e0018 | CTGGGTGGGAGGGTGGGA |
| 69 | c0736 | TGAGGGGCGGGTGGGCCGGGAA |
| 70 | c0604 | TGAGGGGTGGGTGGGGCGGGAA |
| 71 | c0591 | TGAGGGGTGGGAGGGCGGGGAA |
| 72 | c0792 | TGAGGGCAGGGTGGGTCGGGAA |
| 73 | c0515 | TGAGGGGAGGGAGGGAGGGGAA |
| 74 | c0564 | TGAGGGGAGGGCGGGACGGGAA |
| 75 | c0199 | TGAGGGACGGGAGGGTGGGGAA |
| 76 | c0579 | TGAGGGGTGGGAGGGAGGGGAA |
| 77 | c0714 | TGAGGGGCGGGAGGGGTGGGAA |
| 78 | c0967 | TGAGGGCCGGGAGGGTGGGGAA |
| 79 | c0727 | TGAGGGGCGGGTGGGTGGGGAA |
| 80 | c0328 | TGAGGGTTGGGAGGGTCGGGAA |
Table 3
| Serial number: | Title | Sequence |
| 81 | c0499 | TGAGGGTCGGGCGGGAGGGGAA |
| 82 | c0708 | TGAGGGGCGGGAGGGACGGGAA |
| 83 | c0608 | TGAGGGGTGGGTGGGCCGGGAA |
| 84 | c0535 | TGAGGGGAGGGTGGGTGGGGAA |
| 85 | c0596 | TGAGGGGTGGGTGGGACGGGAA |
| 86 | e0024 | CTGGGTGGGTGGGCGGGA |
| 87 | c0160 | TGAGGGAGGGGTGGGCCGGGAA |
| 88 | c0710 | TGAGGGGCGGGAGGGTTGGGAA |
| 89 | c0592 | TGAGGGGTGGGAGGGCCGGGAA |
| 90 | c0706 | TGAGGGGCGGGAGGGATGGGAA |
| 91 | c0528 | TGAGGGGAGGGAGGGCCGGGAA |
| 92 | c0563 | TGAGGGGAGGGCGGGAGGGGAA |
| 93 | c0723 | TGAGGGGCGGGTGGGAGGGGAA |
| 94 | c0211 | TGAGGGACGGGTGGGAGGGGAA |
| 95 | c0179 | TGAGGGAGGGGCGGGAGGGGAA |
| 96 | c0634 | TGAGGGGTGGGCGGGGTGGGAA |
| 97 | c0588 | TGAGGGGTGGGAGGGGCGGGAA |
| 98 | c0467 | TGAGGGTCGGGTGGGAGGGGAA |
| 99 | c0589 | TGAGGGGTGGGAGGGCAGGGAA |
| 100 | c0716 | TGAGGGGCGGGAGGGGCGGGAA |
| 101 | c0522 | TGAGGGGAGGGAGGGGTGGGAA |
| 102 | c0724 | TGAGGGGCGGGTGGGACGGGAA |
| 103 | c0516 | TGAGGGGAGGGAGGGACGGGAA |
| 104 | c0582 | TGAGGGGTGGGAGGGTTGGGAA |
| 105 | c0580 | TGAGGGGTGGGAGGGACGGGAA |
| 106 | c0581 | TGAGGGGTGGGAGGGTAGGGAA |
| 107 | c0590 | TGAGGGGTGGGAGGGCTGGGAA |
| 108 | c0527 | TGAGGGGAGGGAGGGCGGGGAA |
| 109 | c0532 | TGAGGGGAGGGTGGGACGGGAA |
| 110 | e0013 | CTGGGAGGGCGGGAGGGA |
| 111 | e0052 | CTGGGCGGGAGGGCGGGA |
| 112 | c0730 | TGAGGGGCGGGTGGGGTGGGAA |
| 113 | c0521 | TGAGGGGAGGGAGGGGAGGGAA |
| 114 | e0050 | CTGGGCGGGAGGGTGGGA |
| 115 | c0540 | TGAGGGGAGGGTGGGGCGGGAA |
| 116 | c0915 | TGAGGGCGGGGTGGGAGGGGAA |
| 117 | c0570 | TGAGGGGAGGGCGGGGTGGGAA |
| 118 | c0734 | TGAGGGGCGGGTGGGCTGGGAA |
| 119 | t1011 | GGGTGGGAAGGGAGG |
| 120 | c0764 | TGAGGGGCGGGCGGGGCGGGAA |
Table 4
| Serial number: | Title | Sequence |
| 123 | t1113 | GGGAGGGACGGGAGG |
| 124 | c0900 | TGAGGGCGGGGAGGGACGGGAA |
| 125 | c0899 | TGAGGGCGGGGAGGGAGGGGAA |
| 126 | c0766 | TGAGGGGCGGGCGGGCTGGGAA |
| 127 | c0573 | TGAGGGGAGGGCGGGCAGGGAA |
| 128 | t0420 | GGGCGGGAGGGAGGG |
| 129 | t1102 | GCGAGGAAGGGTGGG |
| 130 | c0602 | TGAGGGGTGGGTGGGGTGGGAA |
| 131 | c0585 | TGAGGGGTGGGAGGGGAGGGAA |
| 132 | c0536 | TGAGGGGAGGGTGGGTCGGGAA |
In above-mentioned (b), " one or more " are as long as the polynucleotide such as at above-mentioned (b) have the model of redox active
In enclosing.Above-mentioned " one or more " in any one base sequence of above-mentioned (a) for example, 1～5, preferably 1～3
Individual, more preferably 1 or 2.
In above-mentioned (c), " homogeneity " is as long as such as the polynucleotide at above-mentioned (c) have in the range of redox active
?.Above-mentioned homogeneity for example, more than 80%, more than 85%, preferably more than 90%, more preferably more than 95%, more than 96%,
More than 97%, more preferably more than 98%, particularly preferably more than 99%.Above-mentioned homogeneity can use such as BLAST,
FASTA etc. analyze software and utilize the parameter of acquiescence to calculate (the most same).
In above-mentioned (d), " polynucleotide that can hybridize " are for example, the most mutual with the polynucleotide of above-mentioned (a)
The polynucleotide mended.Above-mentioned hybridization such as can be detected by various hybridization analysis.Above-mentioned hybridization analysis is not particularly limited,
For example, it is also possible to use " the Molecular Cloning: A Laboratory guide second edition (the Molecular Cloning:A of the volumes such as Sambrook
Laboratory Manual2ndEd.) " the method described in [(CSH Press (1989)] etc..
In above-mentioned (d), " stringent condition " can be such as appointing in low stringency condition, middle stringent condition, high stringent condition
A kind of condition of meaning." low stringency condition " for example, 5 × SSC, 5 × Denhardt solution, 0.5%SDS, 50% Methanamide, 32 DEG C
Condition." middle stringent condition " for example, 5 × SSC, 5 × Denhardt solution, 0.5%SDS, 50% Methanamide, the condition of 42 DEG C.
" high stringent condition " for example, 5 × SSC, 5 × Denhardt solution, 0.5%SDS, 50% Methanamide, the condition of 50 DEG C.This area
Technical staff can be by suitably selecting the conditions such as such as temperature, salinity, the concentration of probe and length, ionic strength, time
Set the degree of stringency." stringent condition " can also use " the Molecular Cloning: A Laboratory of the volumes such as Sambrook as escribed above
The guide second edition (Molecular Cloning:A Laboratory Manual2ndEd.) " [(CSH Press
] etc. (1989) condition described in.
The Component units of above-mentioned polynucleotide for example, nucleotide residue.The nucleic acid molecules of the present invention the most only can be enumerated
The DNA being made up of deoxyribonucleotide residues, the DNA etc. containing one or more ribonucleotide residues.The situation of the latter
Under, " one or more " are not particularly limited, such as in above-mentioned polynucleotide for example, 1～3, preferably 1 or 2.
In the nucleic acid molecules of the present invention, above-mentioned polynucleotide are preferably single stranded polynucleotide.Above-mentioned single stranded polynucleotide example
As being preferably able to form stem structure and ring structure by self annealing.Above-mentioned polynucleotide such as be preferably able to formed loop-stem structure,
Interior ring structure and/or projective structure etc..
The nucleic acid molecules of the present invention can be such as double-stranded polynucleotide.Nucleic acid molecules in the present invention is the many nucleoside of double-strand
In the case of acid, such as, in above-mentioned double-stranded polynucleotide, a single stranded polynucleotide is any one polynucleotide above-mentioned, another
Individual single stranded polynucleotide does not limit.Another single stranded polynucleotide above-mentioned can be enumerated such as by many with above-mentioned any one
The polynucleotide that the base sequence of nucleotide complementary is constituted.In the case of the nucleic acid molecules of the present invention is double-stranded polynucleotide,
Above-mentioned double-stranded polynucleotide is made to be dissociated into single stranded polynucleotide by degeneration etc. the most before the use.It addition, dissociate
The above-mentioned single stranded polynucleotide arrived such as can be with stem structure formed as discussed above and ring structure.
In the present invention, " stem structure and ring structure can be formed " such as include being actually formed stem structure and ring structure and
Even if not forming stem structure and ring structure, also being able to form stem structure and the situation of ring structure according to condition." stem knot can be formed
Structure and ring structure " such as include situation about being confirmed by experiment and situation about being predicted by the simulation of computer etc. this
Two kinds of situations.
The nucleic acid molecules of the present invention has redox active, it is therefoie, for example, can be as the succedaneum of oxidoreductase
Use, and can also be applied to utilize as above the detection of fit target substance.
Embodiment
(embodiment 1)
A. condition is investigated
When detection is as the redox reaction of the nucleic acid molecules of evaluation object, its condition is investigated.
(1) immobilization of polynucleotide
As follows, by spacer, known polynucleotide are fixed to commercially available electrochemical detection-type microarray (business
Name of an article CombiMatrix ElectraSense microarray, CombiMatrix company manufacture) electrode on.
Above-mentioned known polynucleotide use the EAD2 (serial number 133) as DNAzyme (with reference to Cheng, X., et
al.(2009)Biochemistry,48,7817-7823.).It addition, as negative control, use is combined with Streptavidin
DNA aptamer SA (serial number 134).
EAD2 (serial number 133)
CTGGGAGGGAGGGAGGGA
SA (serial number 134)
CCGACGCACCGATCGCAGGTTCGG
The length of above-mentioned spacer is set as 0 base length (without spacer), 8 base length, 16 base length, 24 alkali
Base is long.The sequence of above-mentioned spacer is set as poly (dT).
Above-mentioned immobilization is combined on the electrode of 1 above-mentioned microarray by making 3 ' ends of above-mentioned spacer and makes
3 ' ends of above-mentioned known polynucleotide are combined with 5 ' ends of above-mentioned spacer and carry out.It addition, by the length of above-mentioned spacer
Spend 4 kinds of different EAD2 (dT0, dT8, dT16, dT24), the 4 kind SAs different with the length of above-mentioned spacer (dT0, dT8, dT16,
DT24) each 250 are fixed on above-mentioned microarray randomly.
(2) detection of redox reaction
In above-mentioned microarray, interpolation hydrogen peroxide is as substrate, measures in the form of electric current and is produced by redox reaction
The signal of telecommunication.Measure and use determinator (ProductName ElectraSense Reader, CombiMatrix company) (as follows
Sample).Above-mentioned hydrogen peroxide by formed normal concentration (0,1,2,4,8mmol/L) in the way of add in various buffer, and
Add as substrate solution.Above-mentioned buffer uses Tris buffer (pH7.4), Tris buffer (pH8.0), Tris buffering
Liquid (pH8.5), Tris buffer (pH9.0), add the pH after hydrogen peroxide and be adjusted to the value in bracket respectively.
By shown in Figure 1 for their result.Fig. 1 is the figure of the signal of telecommunication under the conditions of representing respectively.In Fig. 1, (A) is pH7.4
Result, (B) is the result of pH8.0, and (C) is the result of pH8.5, and (D) is the result of pH9.0, represents various peroxidating respectively
Result under conditions of hydrogen concentration and spacer lengths.It addition, in each figure, the value of the longitudinal axis represents that the signal of telecommunication of EAD2 (S) is with cloudy
Property comparison signal ratio (S/BG) of the signal of telecommunication (background: BG).As shown in Fig. 1 (A), at pH7.4, concentration of hydrogen peroxide 2mmol/
Under conditions of L, spacer lengths are 24 base length, obtain the highest value.
B. repeatability
Under conditions of identical with above-mentioned " investigation of A. condition ", for 4 kinds of EAD2 that the length of above-mentioned spacer is different
(dT0, dT8, dT16, dT24) each 250, carries out the mensuration of redox reaction.
The results are shown in Fig. 2.Fig. 2 (A) is for each EAD2 and each SA, will measured value measure with second time for the first time
The figure that value is mapped and obtained.Measured value is that the signal measuring value suitable with electric current obtained by said determination device is (as follows
Sample).Transverse axis is first time signal measuring value, and the longitudinal axis represents second time signal measuring value.Fig. 2 (B) is the interval representing each EAD2
The figure of the logarithm of the signal value of section length.In the figure of Fig. 2 (A), the significance test of Pearson product-moment correlation coefficient is shown in the lump
Result.Coefficient R=0.9981756 of Fig. 2 (A).In Fig. 2 (B), about the standard deviation of each spacer lengths, dT0 is
0.09954021, dT8 is 0.09435911, and dT16 is 0.08528754, and dT24 is 0.1027817.Such as Fig. 2 (A) and (B) institute
Show, be to demonstrate the highest repeatability respectively to 4 kinds of each 250 results being measured of EAD2.
C. normality
For the different 4 kinds of EAD2 of the length of the above-mentioned spacer in above-mentioned " investigation of A. condition " (dT0, dT8, dT16,
DT24) each 250 of 4 kinds of SA (dT0, dT8, dT16, dT24) that the measurement result of each 250 is different with the length of above-mentioned spacer
Measurement result, be confirmed whether to defer to normal distribution.It addition, obtain P value by Kolmoforov-Smirnov inspection.
By shown in Figure 3 for their result.Fig. 3 is the relation representing signal value and its frequency for each EAD2 and each SA
Figure.As it is shown on figure 3, can confirm that normal distribution is deferred in the distribution of 250 signals of each EAD2 and each SA.
(embodiment 2)
By being fixed on micro-array chip by the multiple polynucleotide of different Sequence composition, carry out the survey of redox reaction
Fixed.
A. repeatability
As follows, by the spacer being made up of the poly of 24 base length (dT), multiple polynucleotide are fixed to city
On electrode in the micro-array chip (manufacture of trade name CustomArray (registered trade mark) 12K, CombiMatrix company) sold.
For above-mentioned polynucleotide, use as the EAD2 (serial number 133) of DNAzyme, fit SA (serial number 134),
As transcription factor c-Myc gene promoter region partial sequence c-Myc (serial number 135) and as thrombin
Fit TA (serial number 136).
EAD2 (serial number 133)
CTGGGAGGGAGGGAGGGA
SA (serial number 134)
CCGACGCACCGATCGCAGGTTCGG
C-Myc (serial number 135)
TGAGGGTGGGGAGGGTGGGGAA
TA (serial number 136)
GGTTGGTGTGGTTGG
Additionally, use similarly above-mentioned EAD2 (serial number 133), c-Myc (serial number 135) and TA (serial number
136) sequence be changed after change polynucleotide.Prepare 64 kinds of change EAD2 EAD2 being changed and obtain,
1024 kinds of change c-Myc c-Myc being changed and obtain, 1232 kinds of change TA TA being changed and obtain.
Above-mentioned immobilization is combined on the electrode of 1 above-mentioned microarray by making 3 ' ends of above-mentioned spacer and makes
3 ' ends of above-mentioned known polynucleotide or above-mentioned change polynucleotide are combined with 5 ' ends of above-mentioned spacer and carry out.Separately
Outward, using each to EAD2, c-Myc and TA of the comparison as redox active 100, change polynucleotide each 5 solid randomly
Determine on above-mentioned micro-array chip.
Then, the micro-array chip that this is identical is used to carry out the mensuration of 3 redox reaction under the same conditions.Specifically
For, add the Tris buffer (pH7.4) of the hydrogen peroxide containing 2mmol/L, measure in the form of electric current by oxidoreduction
The signal of telecommunication that reaction produces.
The results are shown in Fig. 4.In Fig. 4, first time measured value is mapped with second time measured value and is obtained by (A)
Figure, (B) is figure second time measured value mapped with third time measured value and obtain, and (C) is by first time measured value and the 3rd
The figure that secondary measured value is mapped and obtained.It addition, each figure illustrates the knot of the significance test of Pearson product-moment correlation coefficient in the lump
Really.As shown in Figure 4, in same an array, demonstrate the highest repeatability.
B. normality
Based on the measurement result obtained in above-mentioned " A. repeatability ", (A) 100 c-shown in the figure of Fig. 5 (A)～(D)
The signal value of Myc and the relation of its frequency, signal value and the relation of its frequency, the signal of (C) 100 EAD2 of (B) 100 SA
Value and the relation of its frequency, the signal value of (D) 100 TA and the relation of its frequency.Any one figure all can confirm that signal
Distribution defer to normal distribution.
(embodiment 3)
The micro-array chip of above-described embodiment 2, screening is used to have the new DNAzyme of either high redox activity.
Confirmed to show higher active change polynucleotide than EAD2 by 3 measurement results in above-described embodiment 2.Will
The results are shown in Fig. 6.Fig. 6 is SA, EAD2, c-Myc and TA and the display oxygen of the comparison being denoted as redox active
Change the figure of the signal value of 15 kinds of change polynucleotide of reducing activity.As shown in Figure 6, these change polynucleotide demonstrate ratio
The higher redox active of EAD2.It addition, by T inspection confirm complete these 15 kinds change polynucleotide signal value relative to
EAD2 has significant difference.These the 15 kinds sequences changing polynucleotide are as follows.
Table 5
| Serial number: | Title | Sequence |
| 15 | c0711 | TGAGGGGCGGGAGGGTGGGGAA |
| 22 | c0712 | TGAGGGGCGGGAGGGTCGGGAA |
| 30 | e0032 | CTGGGTGGGCGGGCGGGA |
| 33 | c0605 | TGAGGGGTGGGTGGGCAGGGAA |
| 35 | c0586 | TGAGGGGTGGGAGGGGTGGGAA |
| 39 | c0632 | TGAGGGGTGGGCGGGTCGGGAA |
| 50 | c0584 | TGAGGGGTGGGAGGGTCGGGAA |
| 55 | c0627 | TGAGGGGTGGGCGGGAGGGGAA |
| 65 | c0583 | TGAGGGGTGGGAGGGTGGGGAA |
| 76 | c0579 | TGAGGGGTGGGAGGGAGGGGAA |
| 83 | c0608 | TGAGGGGTGGGTGGGCCGGGAA |
| 90 | c0706 | TGAGGGGCGGGAGGGATGGGAA |
| 97 | c0588 | TGAGGGGTGGGAGGGGCGGGAA |
| 105 | c0580 | TGAGGGGTGGGAGGGACGGGAA |
| 108 | c0527 | TGAGGGGAGGGAGGGCGGGGAA |
Above, the present application is illustrated by reference implementation mode, but the present application is not limited to above-mentioned reality
Execute mode.For composition and the details of the present application, those skilled in the art can be carried out in the range of the present application
It will be appreciated that various changes.
This application requires the priority based on Japanese publication Patent 2011-148562 that on July 4th, 2011 proposes,
The entire disclosure is incorporated in this specification.
Industrial applicability
In accordance with the invention it is possible to easily evaluate the presence or absence of redox active for the nucleic acid molecules as evaluation object
And intensity.It addition, according to the evaluation methodology of such present invention, such as, additionally it is possible to multiple nucleic acids molecule is commented simultaneously
Valency, it is possible to carry out the screening of purpose nucleic acid molecules efficiently.As it has been described above, the nucleic acid molecules with redox active such as may be used
To replace the enzymes such as peroxidase to use, therefore useful in the various fields such as clinical treatment, food, environment.
Claims (6)
1. evaluate the redox active of nucleic acid molecules and screening has the method for nucleic acid molecules of redox active, its
It is characterised by, including:
Detecting step, uses the device electrochemically detecting redox reaction, electrochemically detects by right as evaluating
Elephant nucleic acid molecules catalysis the redox reaction to substrate,
Evaluation procedure, by nucleic acid molecules described in the evaluation of described redox reaction redox active and
Screening step, screening has the nucleic acid molecules of redox active,
Described device has the substrate possessing test section,
Described test section has electrode system,
The described nucleic acid molecules as evaluation object it is configured with on described substrate,
The described nucleic acid molecules as evaluation object is multiple nucleic acids molecule,
Described substrate is the microarray being configured with described multiple nucleic acids molecule,
The described multiple nucleic acids as evaluation object is configured by the bonding pad being made up of the polynucleotide of 20～30 base length
Molecule.
The most described detecting step is for detect by described core in the presence of described substrate
The step of the redox reaction of acid molecule catalysis.
3. method as claimed in claim 1 or 2, wherein, described as be combined with on the nucleic acid molecules of evaluation object can be with
The aptamer that target substance combines.
4. method as claimed in claim 3, wherein, described detecting step is in the presence of described substrate and described target substance
The step of the redox reaction that detection is catalyzed by described nucleic acid molecules.
5. method as claimed in claim 3, wherein,
Described detecting step is included under conditions of there is described substrate and there is not described target substance and detects described oxidoreduction
The step reacted and the step detecting described redox reaction in the presence of described substrate and described target substance,
In described evaluation procedure, for not showing the nucleic acid of described redox active under conditions of there is not described target substance
Molecule, evaluates the described redox active in the presence of described target substance.
6. method as claimed in claim 4, wherein,
Described detecting step is included under conditions of there is described substrate and there is not described target substance and detects described oxidoreduction
The step reacted and the step detecting described redox reaction in the presence of described substrate and described target substance,
In described evaluation procedure, for not showing the nucleic acid of described redox active under conditions of there is not described target substance
Molecule, evaluates the described redox active in the presence of described target substance.
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| PCT/JP2012/066912 WO2013005723A1 (en) | 2011-07-04 | 2012-07-02 | Method for evaluating redox activity of nucleic acid molecule, and nucleic acid molecule having redox activity |
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| US20160169875A1 (en) * | 2012-07-27 | 2016-06-16 | Nec Solution Innovators, Ltd. | Nucleic acid sensor for melamine analysis, device for melamine analysis, and method for melamine analysis |
| WO2014136560A1 (en) * | 2013-03-08 | 2014-09-12 | Ｎｅｃソリューションイノベータ株式会社 | Nucleic acid element candidate molecule and method for screening nucleic acid elements for target analysis using same |
| JP6338191B2 (en) * | 2013-07-01 | 2018-06-06 | Ｎｅｃソリューションイノベータ株式会社 | Attribute estimation system |
| WO2015012060A1 (en) * | 2013-07-23 | 2015-01-29 | Ｎｅｃソリューションイノベータ株式会社 | Sensor for target analysis, device for target analysis, and target analysis method using same |
| WO2015151350A1 (en) * | 2014-03-31 | 2015-10-08 | Ｎｅｃソリューションイノベータ株式会社 | Egg-allergen-binding nucleic acid molecule and use therefor |
| JP7012299B2 (en) * | 2016-04-28 | 2022-01-28 | 国立大学法人東京農工大学 | Increased peroxidase activity aptamer |
| WO2017188426A1 (en) * | 2016-04-28 | 2017-11-02 | 国立大学法人東京農工大学 | Aptamer enhancing peroxidase activity |
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| US6287765B1 (en) * | 1998-05-20 | 2001-09-11 | Molecular Machines, Inc. | Methods for detecting and identifying single molecules |
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| US20140128589A1 (en) | 2014-05-08 |
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| JP5863794B2 (en) | 2016-02-17 |
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